Hfab rhodamine anti gapdh

Cells treated with CREM siRNA or control siRNA were lysed in a radioimmunoprecipitation assay buffer supplemented with protease inhibitors. Samples were normalized for protein concentration, and equal amounts of material in the Laemmli sample buffer were subjected to SDS-PAGE and transferred to nitrocellulose filters. For immunoblotting, the mouse monoclonal anti-CREM antibody (clone 3B; Novusbio, Littleton, CO) and the rabbit monoclonal anti-EWSR1 antibody (ab133288; Abcam, Cambridge, UK) were incubated overnight in a cold room at 1∶1000 dilution in 5% BSA/TBS/Tween 0.1%. After that, the membrane was incubated for 1 hr at room temperature with a mix of goat anti-rabbit StarBright Blue 520 and goat anti-mouse StarBright Blue 700, and hFAB rhodamine anti-GAPDH (Bio-Rad, Hercules, CA, USA) antibodies at 1:1000 dilution. Equal protein loading was evaluated by the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The membrane was washed 3× with TBST between incubations. Bound proteins were detected by fluorescence using ChemiDoc Gel Imaging System (Bio-Rad).

Cardiac tissue was cracked by RIPA and phosphatase inhibitor (Bimake, B15001) mixture to extract protein. Protein concentration was determined by BCA protein reagents (Thermo scientific, no. 23227). 20 μg of proteins were resolved on SDS-PAGE, transferred to a low-fluorescence PVDF membrane (Thermo scientific, no. 22860), and incubated with the following primary antibodies: anti-A2b (1 : 1000, GeneTex54903), antiphospholamban (PLB, 1 : 5000, Abcam2865), antiphospholamban phosphor S16 (P-PLB, 0.5 μg/ml, Abcam15000), antiryanodine receptor 2 (RyR 2, 1 : 5000, Abcam2861), NCX1 (1 : 1000, Abcam177952), SERCA2a (1 : 1000, Abcam2861), troponin C (1 : 4000, Abcam137130), and troponin I (1 : 2000, Abcam10231). All of the membranes were incubated at 4°C overnight. After incubation with fluorescent secondary antibodies (1 : 15000, Licor, IRDye® 800CW, IRDye® 680RD) for 1 h at room temperature, the membrane was washed 3 times and incubated with the housekeeping protein HFAB™ Rhodamine anti-GAPDH (1 : 1000, Bio-rad 12004167) for 1 h at room temperature. Images were taken with a Typhoon FLA9500 (GE Healthcare) and analyzed by using ImageQuant TL software.